Fusion polypeptide for detection of conserved combinatorial or composite epitopes in non-conserved proteins

ABSTRACT

The present invention provides multiepitope-binding fusion polypeptides for use in a method for the detection of the presence of human immunodeficiency virus, HIV, in a biological sample. The present invention also provides a method for producing multiepitope-binding fusion polypeptides.

FIELD OF THE INVENTION

The present invention provides a fusion polypeptide for reliablediagnostic detection of highly variable proteins that lack conservedantibody binding epitopes. This is achieved via recognition ofindependent but linked structural determinants in the same targetprotein using polypeptides comprised of two or more binding units. Thestructural determinants targeted with this approach consist of short(3-5 residues) conserved amino acid sequences in these otherwisevariable proteins. These tri-, tetra-, and pentapeptides can beefficiently targeted using bioengineered high affinity bindingpolypeptides (BHAP), but since such short sequences may appear in manyother proteins in the specimens of interests, e.g. serum samples, thediagnostic value of this binding is of limited value. However,cooperative binding of multiepitope-binding fusion polypeptides (MEBIP)comprised of two or more BHAPs that bind to multiple conserved tri-,tetra-, or pentapeptides can be used to overcome this problem, and toensure specific targeting of the protein of interest with a superioraffinity that enables diagnostic detection of the said protein.Recombinant antibody fragment is one example of several types of bindingproteins that could represent one or all BHAP subunits in a MEBIP. As aconsequence, this invention provides a novel means for reliable andquantitative detection of proteins encoded by viruses with highmutational capacities, such as HIV.

BACKGROUND OF THE INVENTION

Schupbach et al. (Journal of Medical Virology, 2001, 65:225-232)discloses that heat-denatured, amplification-boosted p24 antigen can beused as an alternative to HIV RNA testing in order to monitor thetreatment of HIV infection. Respess et al. (Journal of ClinicalMicrobiology, 2005, 43(1):506-508) and Knuchel et al. (Journal ofClinical Virology, 2006, 36:64-67) also disclose ultrasensitive p24antigen assays as an alternative to HIV RNA testing.

Boder et al. (PNAS, 2000, 97(20):10701-10705) discloses directedevolution of antibody fragments with monovalent femtomolarantigen-binding affinity. Holliger and Hudson (Nature Biotechnology,2005, 23(9):1126-1136) reviews engineered antibody fragments. Nygren andUhlen (Current Opinion in Structural Biology, 1997, 7:463-469) and Hosseet al. (Protein Science, 2006, 15:14-27) review engineering of proteindisplay scaffolds for molecular recognition.

Binz et al. (Nature Biotechnology, 2005, 23(10):1257-1268) and Hey etal. (Trends in Biotechnology, 2005, 23(10):514-422) review engineeringof novel binding proteins from nonimmunoglobulin domains.

Bi-specific recombinant antibody molecules that can recognize and bringtogether two different ligands are well-known in the literature (seee.g., Albrecht et al., J Immunol Meth 310: 100-16, 2006). Bi-specificrecombinant antibodies that bind to two different epitopes in the sameprotein have also been described (see e.g. Neri et al., J Mol Biol 246:367-73, 1995; Zhou, J Mol Biol 329: 1-8, 2003). The combinatorialbinding resulted in a significant increase in binding affinity comparedto binding of each of the two recombinant antibodies alone. While MEBIPsmay have similarities with construction of bi-specific recombinantantibodies, it is important to appreciate that the present innovation isnovel and unrelated to the described design and use of bi-specificrecombinant antibodies.

Although helpful, the increased affinity involved in cooperative bindingof more than one covalently joined BHAP (whether a recombinantantibodies or another type of molecule) is not the reason for targetingmultiple regions in the protein of interest. Instead, the key idea ofthis innovation is to combine scattered short conserved peptides withina variable protein to a “virtual epitope” that provides sufficientcomplexity for diagnostic specificity in detection. To provide suchstructural complexity a linear peptide epitope should consist of atleast six residues. However, a look into available sequence databasesshows that well conserved continuous 6-residues amino acids stretchesare hard to find in many highly variable microbial proteins, inparticular those of RNA viruses. For example, the HIV-1 p24 protein doesnot contain a single hexapeptide (6-mer) that would be conserved in morethan 99% of known HIV-1 sequences, which makes its reliableimmunological detection problematic. As discussed below, coordinateddetection of combinations of conserved tri-, tetra-, or pentapeptidesusing the MEBIP approach can help to solve this problem. Thus, thisnovel approach therefore allows development of better means fordiagnostic detection of highly variable microbial proteins, such asHIV-1 p24.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Amino acid sequence of p24 protein of a representative HIV-1strain (SEQ ID NO:1). The FIGURE shows relative conservation of theresidues of p24 among clades A-K and various circulating recombinantviruses of the predominant M-type of HIV-1 as well as O- and N-typeviruses and related SIV viruses from chimpanzees. Score of 1 indicatesconservation of more than 99.75%, score of 2 indicates conservationof >99.50%, score of 3 indicates conservation of >99.00%, score of 4indicates conservation of >98.00%, and score of 5 indicates conservationof >97.00% (the score is shown above each residue). Residues that areless than 97% conserved are not scored. Continuous peptide stretcheswith an overall conservation of >99.00% are underlined and indicated inboldface.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided for some terms used in thisspecification.

“Antibody” in its various grammatical forms is used herein as acollective noun that refers to a population of immunoglobulin moleculesand/or immunologically active portions of immunoglobulin molecules,i.e., molecules that contain an antigen binding site or a paratope.

An “epitope” is the part of a macromolecule, such as polypeptides, thatis recognized by the immune system, specifically by antibodies, B cells,or cytotoxic T cells. Most epitopes recognized by antibodies can bethought of as three-dimensional surface features of an antigen molecule.These features fit precisely and thus bind to antibodies. These surfacesmay depend on tertiary protein structure, such that residues that forman epitope are positioned apart from each other in the amino acidsequence of a protein (conformational epitopes), or may be formed bycontinuous peptide regions within a protein (linear epitopes).Therefore, if a protein is denatured, as often is the case in diagnosticuse of antibodies, only linear epitopes can be used for detection. Thenumber of consecutive amino acid residues that form a linear epitoperecognized by an antibody varies, but typically ranges from six to ten(6-10). However, natural antibodies can recognize shorter epitopes withsignificant affinities, and recombinant antibodies can be targeted tobind even to a single amino acid residue.

An “antigen-binding site”, a “paratope”, is the structural portion of anantibody molecule that specifically binds an antigen.

“Single-chain antibody” (scFv) is used to define a molecule in which thevariable domains of the heavy and light chain of an antibody are joinedtogether via a linker peptide to form a continuous amino acid chainsynthesised from a single mRNA molecule (transcript).

“Immunoassay” is a biochemical test that measures the level of asubstance in a biological liquid, typically serum, plasma, urine, orother body fluids, using the reaction of an antibody or antibodies toits antigen. The assay uses the specific binding of an antibody to itsantigen. Monoclonal antibodies are often used because they usually bindto a single site of a molecule to be detected, and therefore providemore specific and accurate testing, which is not interfered by othermolecules in the sample. The antibodies used must have a high affinityfor the antigen. The presence of the antigen can be measured forinstance in the diagnosis of infectious diseases by detecting themicrobe specific molecular structures. Detecting the quantity of theantigen can be achieved by a variety of methods. One of the most commonused techniques is to label the antigen or antibody. The label mayconsist of an enzyme (Enzyme ImmunoAssay, EIA), fluorescence (FIA),luminescence (LIA) or they can be based on agglutination, nephelometry,turbidimetry or immunoblotting (Western Blot).

Immunoassays can be either competitive or non-competitive, and they canbe homogeneous or heterogeneous. In a competitive assay, the antigen inthe sample competes with the labelled antigen to bind with antibodies.The amount of labelled antigen bound to the antibody site is thenmeasured. The response will be inversely proportional to theconcentration of antigen in the sample, because the greater theresponse, the less antigen in the sample is available to compete withthe labelled antigen.

In non-competitive immunoassays, often referred to as “sandwich assay”,antigen in the sample is bound to the “capture” antibody and the amountof the labelled antibody on the site is measured. Unlike in the case ofcompetitive assay the result will be directly proportional to theconcentration of the antigen.

A heterogeneous immunoassay will require an extra step to remove unboundantibody or antigen from the site, usually using a solid phase material.Homogenous assays do not require the separation phase to remove theunbound antibody or antigen molecules. Immunoassays have a particularlyimportant role in the diagnosis of HIV.

The abbreviation “MEBIP” refers to “multiepitope-binding fusionpolypeptides”, which are genetically engineered protein constructscomprising two or more independent binding units that bind to differentsites in a common target protein. One or more of the binding unitswithin a MEBIP may be a scFv.

The “virtual epitope” is a structure typically formed by two stretchesof three to five amino acid residues that tend to be constant even inotherwise highly variable proteins, such as many viral proteins, and canserve as a ligand for a MEBIP. “Virtual epitope” may overlap with anantigenic epitope, but may not be targeted by a traditional antibody.

As used herein, the term “specifically binding”, or “specificallyrecognizing”, or the expression “having binding specificity to anepitope” refers to a low background and high affinity binding between aMIEBIP or a fragment or derivative thereof and its target molecule (i.e.lack of non-specific binding). In other words, the terms (and equivalentphrases) refer to the ability of a binding moiety (e.g., a receptor,antibody, ligand or antiligand) to bind preferentially to a particulartarget molecule (e.g., ligand or antigen) in the presence of aheterogeneous population of proteins and other biologics (i.e., withoutsignificant binding to other components present in a test sample).Typically, specific binding between two entities, such as a ligand and areceptor, means a binding affinity of at least about 10⁶ M⁻¹, andpreferably at least about 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹, more preferably atleast about 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ M⁻¹.

The terms “specificity” or “high specificity” may also refer to thecapacity of a binding polypeptide, such as MEBIP, to bind to 95%, 95.5%,96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% of the variantsof its non-conserved polypeptide ligand.

The terms “biopanning” and “phage display library” are used herein inthe same way as in the US Patent Application No. 2005/0074747 (Arap etal.).

Further, the classic definition of an antigen is “any foreign substance”that elicits an immune response (e.g., the production of specificantibody molecules) when introduced into the tissues of a susceptibleanimal and is capable of combining with the specific antibodies formed.Antigens are generally of high molecular weight and commonly areproteins or polysaccharides. Polypeptides, lipids, nucleic acids andmany other materials can also function as antigens. Immune responses mayalso be generated against smaller substances, called haptens, if theseare chemically coupled to a larger carrier protein, such as bovine serumalbumin, keyhole limpet hemocyanin (KLH) or other synthetic matrices. Avariety of molecules such as drugs, simple sugars, amino acids, smallpeptides, phospholipids, or triglycerides may function as haptens. Thus,given enough time, just about any foreign substance will be identifiedby the immune system and evoke specific antibody production. However,this specific immune response is highly variable and depends much inpart on the size, structure and composition of antigens. Antigens thatelicit strong immune responses are said to be strongly immunogenic.

Characteristics of a good antigen include:

-   -   Areas of structural stability and chemical complexity within the        molecule.    -   Significant stretches lacking extensive repeating units.    -   A minimal molecular weight of 8,000-10,000 Daltons, although        haptens with molecular weights as low as 200 Da have been used        in the presence of a carrier protein.    -   The ability to be processed by the immune system.    -   Immunogenic regions which are accessible to the antibody-forming        mechanism.    -   Structural elements that are sufficiently different from the        host.    -   For peptide antigens, regions containing at least 30% of        immunogenic amino acids: K, R, E, D, Q, N.    -   For peptide antigens, significant hydrophilic or charged        residues.

Because an antibody binding epitope can consist of only a few aminoacids it may not have any diagnostic value, because the same epitope maybe present in many other proteins in the same specimen (e.g. a serumsample). Thus, an epitope useful, e.g., in detection of microbialproteins in human blood should not be present in human serum, which hasbeen estimated to include up to 10,0000 different proteins. Whenassuming that the average size of a serum protein would be 50 kD(approximately 500 amino acids), and that all 20 natural amino acidswould be equally used in human proteins, the likelihood of any given di-tri- or tetrapeptide to be found in serum can be calculated to beessentially 100%. The corresponding likelihood of a given pentapeptide(5 amino acids) to be present among a set of 10,000 hypothetical 50 kDproteins is 79%, and the likelihood of a hexapeptide (6 amino acids)being present is 7.5%.

Thus, it can be estimated that the diagnostic utility of 79% of all5-mer and 7.5% of all 6-mer antibody epitopes in microbial pathogens iscompromised by their presence in normal serum. In other words, only 21%antibodies (or other types of specific binding proteins) capable ofbinding sufficiently tightly to a microbial protein via recognizing a5-mer linear peptide epitope can be expected not to bind to an epitopepresent in human serum and thus be useful for diagnostic detection ofthis microbe. By comparison, the great majority of antimicrobialantibodies that recognize linear epitope consisting of six or moreresidues does not have this problem. Of course, the utility ofdiagnostic antibodies may also be compromised because they cross-reactwith epitopes that are encoded by unrelated peptide sequences, but thelength of the target epitope is less relevant in the case of thiscomplication.

Based on the above calculations, it is evident that linear epitopesconsisting of five residues have only limited value, and epitopesshorter than five residues are not useful as detection targets inmicrobial diagnostics. However, the situation is different if thecombinatorial presence of such short epitopes in a single protein isconsidered. The calculated likelihood of appearance of differentcombinations of tri-, tetra-, and pentapeptides in a single proteinamong 1000 or 10,000 hypothetical 500 residue-long polypeptides is shownbelow (Table 1).

TABLE 1 Chance of Chance of being present being present in 1000 50 kD in10,000 50 kD Epitope combination proteins proteins Tripeptide +tripeptide 97% ~100%   Tripeptide + tetrapeptide 17%  85% Tripeptide +pentapeptide 0.94%   9.0% Tetrapeptide + tetrapeptide 1.0%  9.3%Tetrapeptide + pentapeptide 0.05%   0.5% Pentapeptide + pentapeptide ~0%0.02% 

This analysis shows that epitopes as short as three or four residuescould be very useful for diagnostic purposes if their combined presencewith another epitope of suboptimal length (tetra- or pentamers) could bedetected. Together such short and thereby non-diagnostic epitopes couldbe considered as diagnostically valuable “virtual epitopes”. Thisconcept could be highly useful when trying to reliably detect highlyvariable microbial proteins, such as HIV p24 antigen, which contain fewamino acid residues that would be positioned next to each other andconserved in most viral strains and quasispecies.

Based on the above, the present invention provides a method forproducing a fusion polypeptide, i.e. a MEBIP, capable of specificallybinding simultaneously to at least two epitopes of a polypeptide antigenknown to be variable, said continuous epitopes consisting of 3, 4 or 5adjacently positioned conserved amino acid residues of said antigen, themethod comprising the steps of:

a) selecting of 3 to 5 amino acid long conserved regions in the antigenby computational analysis of known amino acid sequences of the antigen;b) preparing a peptide based on the selected conserved region of theantigen;c) contacting a library of particles expressing binding proteins, suchas a phage library of single chain antibodies, with said peptide;d) isolating those particles which express binding proteins havingbinding activity towards said peptide;e) subjecting nucleic acid obtained or derived from the particle(s)isolated in step d) to mutagenesis;f) preparing a library of particles expressing binding proteins based onthe particles obtained from step e);g) contacting a library obtained from step f) with said peptide orfragment thereof;h) isolating those particles which express binding proteins havingimproved binding activity towards said peptide or a fragment thereof;i) repeating steps e) to h) one or more times;j) obtaining particles which are able to specifically bind to an atleast 3 to 5 adjacent or non-contiguous amino acids long epitope in saidantigen from the particles obtained from step i);k) preparing said fusion polypeptide based on the particles isolated instep j) by combining into one fusion polypeptide the binding specificityof two of said particles having specificity to at least two differentepitopes of said antigen resulting in a fusion polypeptide having highspecificity with regard to variants of said antigen.

Preferably the MEBIP obtained in step k) specifically binds to 95%,95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or more than 99% of thevariants of said antigen, which preferably is the p24 polypeptide ofHIV.

Preferably said peptide in step b) is selected from 3 to 5 amino acidlong regions of the conserved peptides of the p24 polypeptide of HIVconsisting of: NAWVK, FRDY, RAEQ, NPDC, VGGP, AW Vii, NAW V, RDY, FRD,AEQ, RAE, PDC, NPD, GGP, VGG, WVK, AWV, NAW, SDI, PVG, GLN, WMT, TLL,EMM, and HKA.

The present invention is also directed to a fusion polypeptide, i.e. aMEBIP, capable of specifically binding simultaneously to at least twoepitopes of a polypeptide antigen known to be variable, said epitopesconsisting of 3 to 5 residues long stretches of conserved amino acidresidues of said antigen, and said polypeptide having high specificitywith regard to variants of said antigen. Such MEBIP can be obtained bythe method described above.

HIV Assay

In the case of detection of human immunodefiency virus, HIV, the problemis that the antigenic sites of the virus are constantly and rapidlychanging. The solution of the present invention is to provide means toprepare a MEBIP, which specifically binds to two different amino acidstretches consisting of highly conserved 3 to 5 amino acid residues longepitopes of the p24 polypeptide, which would be difficult or impossibleto accomplish with conventional antibodies. The MEBIPs thus obtained canbe used in detection methods in the same way as antibodies and are thususeful in detecting the presence of human immunodeficiency virus in abiological sample.

A person skilled in the art can easily apply the above approach also toother antigen assays. Thus, the present invention provides a generalmethod for detecting the presence of an antigen in a biological sample,the method comprising

a) contacting said sample or a fraction thereof with a MEBIP; andb) detecting a complex of said polypeptide and antigen, the presence ofsaid complex indicating the presence of said antigen in said sample.

Preferably, said antigen is the p24 polypeptide of HIV and the method isfor the detection of HIV in the sample.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, to provide additionaldetails with respect to its practice, are incorporated herein byreference. The present invention is further described in the followingexamples, which are not intended to limit the scope of the invention.

EXAMPLES Example 1

To identify conserved regions useful as virtual epitopes in HIV p24 alarge number of individual amino acid sequence available in publicdatabases, such as http://www.hiv.lanl.gov/content/index, were alignedwith each other, and the relative conservation of each amino acidresidue was evaluated. Based on this analysis peptide stretchesconsisting of three or more residues conserved in more than 99% of thesequences were selected for further analysis (see FIG. 1).

Synthetic peptides containing one or several potential conserved regionsfor virtual epitopes in p24 of HIV, are used to screen large librariesof polypeptides that can serve as MEBIP precursors using affinity basedselection methods. For example, the ETH-2-Gold phage display librarygenerated by Neri and colleagues (Proteomics 5:2340-2350, 2005)containing three billion individual recombinant antibody clones isscreened for polypeptides that can specifically interact with conservedregions-containing peptides. Several libraries containing potentialligand binding polypeptides based on non-Ig-derived polypeptides alsoexist (see e.g. Nature Biotechnology 23:1257-1268, 2005) or can bedesigned de novo, and are used to screen for polypeptides as to developMEBIPs. In addition to screening of such MEBIP precursor libraries withsynthetic peptides, recombinant proteins containing one or morepotential conserved regions, as well as denatured HIV capsid proteins(p24) are used as ligands in affinity selection. In the latter casetargeting of the binding to peptides with said conserved regions can beachieved for example via use of these peptides to elute phages withdesired binding specificities.

Following generation of potential MEBIP molecules that bind to thesepeptides, for example by screening scFv phage libraries (basicprinciples of screening recombinant antibody libraries are reviewed byHoogenboom, Nature Biotechnology 23(9):1105-1116), the residues thataccount for this binding are confirmed using peptide array technology.Any combination of two or more of the epitopes shown in FIG. 1 is apotential combinatorial target for MEBIP binding to be used in detectionof HW p24.

Example 2

MEBIP precursors that bind both to denatured p24 as well as a definedvirtual epitope-containing peptide are chosen for further development.Binding affinity of these pre-MEBIPs is maximized via reiteratedmutagenesis and affinity selection, as described by the inventors intheir previous studies related to SCA engineering (Biochemistry41:12729-12738, 2003). Both random mutagenesis using error-prone PCR asdescribed in Biochemistry article cited above or other similartechniques, as well as targeted mutagenesis of the binding surfaces inthe pre-MEBIPs, or combinations of these approaches are used.Traditional phage-display based on the M13-derived phagemid plus helperbacteriophage-mediated approach are used for affinity selection andamplification of the improved pre-MEBIP molecules, but other relatedscreening methods can also be used.

Subsequently, a MEBIP is prepared (see e.g., Albrecht et al., 2006,Journal of Immunological Methods 310:100-116) by combining into onefusion polypeptide the binding specificity of two of the pre-MEBIPshaving specificity to conserved regions of a virtual epitope in p24resulting in a fusion polypeptide, a MEBIP. Finally, binding affinity ofthe assembled MEBIP towards its composite target is further optimizedusing the same methodology as initially used to engineer bindingproperties of the pre-MEBIP subunits.

The binding affinities and other salient properties are thencharacterized in detail. The properties of optimal MEBIPs, which areused as such or as various fusion protein derivatives for building ofnovel p24 detection assays include: 1) High affinity for heat-denaturedHIV p24 protein, preferably meaning a dissociation constant lower than10⁻¹²M, 2) absolute conservation of the virtual epitope in more than 99%of the relevant virus strains, and 3) good solubility and ease oflarge-scale recombinant production.

1. A fusion polypeptide specifically binding simultaneously to at leasttwo epitopes of a polypeptide antigen known to be variable, saidepitopes consisting of 3 to 5 adjacently positioned conserved amino acidresidues of said antigen and said binding resulting in high specificityand broad coverage of said fusion polypeptide in binding to variants ofsaid antigen.
 2. The fusion polypeptide according to claim 1, whereinsaid epitopes together consist of at least 8 conserved amino acidresidues of said antigen.
 3. The fusion polypeptide according to claim1, wherein said fusion polypeptide specifically binds to 95%, 95.5%,96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% of the variantsof said antigen.
 4. The fusion polypeptide according to claim 1comprising two single chain antibodies covalently linked to each other.5. The fusion polypeptide according to claim 1, wherein the epitopesconsist of 3 to 4, or 4 to 5 adjacent amino acid residues.
 6. The fusionpolypeptide according to claim 5, wherein one or both epitopes consistof 3, 4, or 5 adjacent amino acid residues.
 7. The fusion polypeptideaccording to claim 1, wherein said polypeptide has affinity of 10⁻¹² to10⁻¹⁵ M to the antigen.
 8. The fusion polypeptide according to claim 1,wherein the antigen is the p24 polypeptide of HIV and the epitopes areselected from the 3 to 5 amino acid long regions of the p24 polypeptideof HIV consisting of: NAWVK, FRDY, RAEQ, NPDC, VGGP, AWVK, NAWV, RDY,FRD, AEQ, RAE, PDC, NPD, GGP, VGG, WVK, AWV, NAW, SDI, PVG, GLN, WMT,TLL, EMM, and HKA.
 9. The fusion polypeptide according to claim 1,wherein said fusion polypeptide is obtained by subjecting a bindingpolypeptide to successive rounds of biopanning.
 10. The fusionpolypeptide according to claim 9, wherein said biopanning is based onphage display systems.
 11. The fusion polypeptide according to claim 1,wherein the epitope is not immunogenic.
 12. The fusion polypeptideaccording to claim 1, wherein said fusion polypeptide is labelled. 13.Method for producing a fusion polypeptide capable of specificallybinding simultaneously to at least two epitopes of a polypeptide antigenknown to be variable, said epitopes consisting of 3 to 5 adjacentconserved amino acid residues of said antigen, the method comprising thesteps of: a) selecting 3 to 5 amino acid long conserved regions in theantigen by computational analysis of known amino acid sequences of theantigen; b) preparing peptides based on the selected conserved regionsof the antigen; c) contacting a library of particles expressing bindingproteins with one or more of said peptides; d) isolating those particleswhich express binding proteins having binding activity towards thepeptides; e) subjecting nucleic acid obtained or derived from theparticle(s) isolated in step d) to mutagenesis; f) preparing a libraryof particles expressing binding proteins based on the particles obtainedfrom step e); g) contacting a library obtained from step f) with one ormore of said peptides or fragment thereof; h) isolating those particleswhich express binding proteins having improved binding activity towardssaid peptides or a fragment thereof; i) repeating steps e) to h) one ormore times; j) obtaining particles which are able to specifically bindto an at least 3 to 5 adjacent amino acids long epitope in said antigenfrom the particles obtained from step i); k) preparing said fusionpolypeptide based on the particles isolated in step j) by combining intoone fusion polypeptide the binding specificity of two of said particleshaving specificity to at least two different epitopes of said antigenresulting in a fusion polypeptide having high specificity with regard tovariants of said antigen.
 14. The method according to claim 13, whereinsaid fusion polypeptide obtained in step k) specifically binds to 95%,95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% of thevariants of said antigen.
 15. The method according to claim 13, whereinsaid library is a phage library of single chain antibodies.
 16. Themethod according to claim 13, wherein said antigen is the p24polypeptide of HIV.
 17. The method according to claim 16, wherein saidpeptide is selected from the 3 to 5 amino acid long regions of the p24polypeptide of HIV consisting of: NAWVK, FRDY, RAEQ, NPDC, VGGP, AWVK,NAWV, RDY, FRD, AEQ, RAE, PDC, NPD, GGP, VGG, WVK, AWV, NAW, SDI, PVG,GLN, WMT, TLL, EMM, and HKA.
 18. The method according to claim 13,wherein the epitopes consist of 3 to 4, or 4 to 5, adjacent amino acidresidues.
 19. The method according to claim 13, wherein one or bothepitopes consist of 3, 4, or 5 adjacent amino acid residues.
 20. Themethod according to claim 13, wherein said fusion polypeptide hasaffinity of 10⁻¹² to 10⁻¹⁵ M to the antigen.
 21. A fusion polypeptidecapable of specifically binding simultaneously to at least two epitopesof a polypeptide antigen known to be variable, said epitopes consistingof 3 to 5 adjacent conserved amino acid residues of said antigen, andsaid polypeptide having high specifity with regard to variants of saidantigen, obtained by the method according to claim
 13. 22. The fusionpolypeptide according to claim 21, wherein said fusion polypeptidespecifically binds to 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%,99%, 99.5% or 100% of the variants of said antigen.
 23. Method fordetecting the presence of an antigen in a biological sample, the methodcomprising a) contacting said sample or a fraction thereof with a fusionpolypeptide according to claim 1; b) detecting a complex of saidpolypeptide and antigen, the presence of said complex indicating thepresence of said antigen in said sample.
 24. The method according toclaim 23, wherein said polypeptide is the polypeptide according to claim8, said antigen is the p24 polypeptide of HIV and the method is for thedetection of HIV in the sample.